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Acute Eye Irritation

Objective/Purpose

To assess the eye irritation / corrosion potential of pesticide.

Animal welfare Approach for Eye Irritation test

In view of animal welfare concerns, prior to the test for acute eye irritation/corrosion, a weight-of-the-evidence analysis should be performed on the existing relevant data. Where insufficient data are available, it is recommended that they be developed through application of sequential testing.

In addition, the findings of dermal irritation/corrosion test is to be considered to predict eye corrosion prior to undertaking detail eye irritation test. Materials which have demonstrated definite corrosion or severe irritation in a dermal study, need not be further tested for eye irritation.

1.                  Principle of  the Test

The test article is instilled in a single dose to one eye of the test animal; untreated eye serves as a control. The eyes are examined at regular intervals for irritation/corrosion; and their reversibility.  Other effects in the eye and adverse systemic effects are also observed for complete evaluation  of effects. Moribund or severely distressed animals are humanely killed. 

 

2.                  Test Animals

Selection of Animal Species: The albino rabbit is the preferred animal species.

3.                  Age

Healthy young adult animals (not older than 2 years and having no ocular abnormality in ophthalmological examination of eye) are to be included in the study.

4.                  Housing & Feeding Conditions

 

Temperature - 22±3ºC

Relative Humidity – 50-60% (However, humidity should not be below 30% or exceed                                 70%  at any given point of time).

Lighting  –  12 hours light and dark cycle

Diet and water – Standard laboratory diet specific to the species and  filtered water, free                                  from contamination

 

 

 

5.                  Acclimatization

 

The animals should be acclimatized to the laboratory conditions for at least 5 days by keeping in individual cages prior to dosing.

 

6.                  Application Method

 

Before inducting the animals in the experiment, both the eyes of all test animals are to be examined within 24 hrs. before the start of test. The substance to be tested should be placed in a single dose in the conjunctival sac to one of the eyes of the experimental animal; the untreated eye serves as the control.

 

7.                  Number  of animals

 

Maximum 3 animals. Initial test is to be performed on one animal followed by confirmatory test in 1-2 additional animals. If a corrosive or severe irritant effect is observed in the initial test, no further testing to be undertaken.  If a corrosive effect is not observed then irritant or negative response is to be confirmed by testing in sequential manner in one animal at a time, rather than exposing the two additional animals simultaneously. If the second animal reveals corrosive or severe irritant effects, the test is not continued. In case of weak or moderate irritant responses on 2nd animal  it can be confirmed on 3rd rabbit.

 

8.                    Dose Levels

 

Liquids:  A dose of 0.1 ml ;

Solids, pastes:  a volume of 0.1 ml or a weight of not more than 100 mg.

 

Note: If the test substance is solid or granular, grind it to a fine dust.  Local anesthetics may be used on a case-by-case basis. If the weight-of-the-evidence analysis indicates that the substance has the potential to cause pain, or initial testing shows that a painful reaction will occur, a local anesthetic may be used prior to instillation of the test substance. The type, concentration, and dose of the local anesthetic should be carefully selected to ensure that differences in reaction to the test substance will not result from its use. The control eye should be similarly anaesthetized.

 

9.                  Exposure duration

 

The eyes of the test animals should not be washed for at least 24 hours following instillation of the test substance, except for solids and in case of immediate corrosive or irritating effects where it should be washed after 1 hour.

 

If   required, for   some   substances   shown   to   be   irritating   by   this   test,   additional testing may be indicated using animals where eyes are washed soon after instillation of the substance.

 

Half a minute after instillation, the eyes of the animals are washed with water for 30 sec, using a volume and velocity of flow which should not cause injury. However, this is optional.

 

 

10.              Observations and Examination

 

The eyes should be examined at 1, 24, 48, and 72 hours as per the prescribed scale  given below w.r.t grading of ocular lesions  (0-4) on Cornea, Iris, Conjunctivae. 
If animals do not develop ocular lesions during first 3 days after instillation   then study can be terminated.  Any other effect/lesion in the eye (e.g. Staining, pannus etc.) or adverse systemic effect should be recorded.

 

Animals with mild to moderate lesions should be observed until the lesions clear or for 21 days in order to determine their reversibility or irreversibility.
Animals with severe pain or distress due to severe eye lesions known to be irreversible, should be humanely killed without delay and the substance assessed accordingly.

 

 

11.              Result Assessment

 

 

 

GRADING OF OCULAR LESIONS

 

 

Cornea

Opacity: degree of density (readings should be taken from most dense area)*

 

No ulceration or opacity...................................................................................................................0

Scattered or diffuse areas of opacity (other than slight dulling of normal lustre);

details of iris clearly visible ............................................................................................................ 1

Easily discernible translucent area; details of iris slightly obscured ...............................................2

Nacrous area; no details of iris visible; size of pupil barely discernible .........................................3

Opaque cornea; iris not discernible through the opacity .................................................................4

 

Maximum possible: 4

* The area of corneal opacity should be noted

 

 

Iris

Normal...............................................................................................................................................0

Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia;

or injection; iris reactive to light (a sluggish reaction is considered to be an effect ..........................1

Hemorrhage, gross destruction, or no reaction to light ......................................................................2

Maximum possible: 2

Conjunctivae

Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris)

Normal................................................................................................................................................0

Some blood vessels hyperaemic (injected) .........................................................................................1

Diffuse, crimson colour; individual vessels not easily discernible .....................................................2

Diffuse beefyred.................................................................................................................................3

Maximum possible: 3

Chemosis

Swelling (refers to lids and/or nictating membranes)

Normal..................................................................................................................................................0

Some swelling above norma .................................................................................................................1

Obvious swelling, with partial eversion of lids.....................................................................................2

Swelling, with lids about half closed ....................................................................................................3

Swelling, with lids more than half closed .............................................................................................4

Maximum possible: 4

 

 

 

 

12.              Toxicity  endpoint

 

Irritation or corrosion of eye